Exploring Microorganisms Associated to Acute Febrile Illness and Severe Neurological Disorders of Unknown Origin: A Nanopore Metagenomics Approach.

Acute febrile illness (AFI) and severe neurological disorders (SNDs) often present diagnostic challenges due to their potential origins from a wide range of infectious agents. Nanopore metagenomics is emerging as a powerful tool for identifying the microorganisms potentially responsible for these undiagnosed clinical cases. In this study, we aim to shed light on the etiological agents underlying AFI and SND cases that conventional diagnostic methods have not been able to fully elucidate. Our approach involved analyzing samples from fourteen hospitalized patients using a comprehensive nanopore metagenomic approach. This process included RNA extraction and enrichment using the SMART-9N protocol, followed by nanopore sequencing. Subsequent steps involved quality control, host DNA/cDNA removal, de novo genome assembly, and taxonomic classification. Our findings in AFI cases revealed a spectrum of disease-associated microbes, including Escherichia coli, Streptococcus sp., Human Immunodeficiency Virus 1 (Subtype B), and Human Pegivirus. Similarly, SND cases revealed the presence of pathogens such as Escherichia coli, Clostridium sp., and Dengue virus type 2 (Genotype-II lineage). This study employed a metagenomic analysis method, demonstrating its efficiency and adaptability in pathogen identification. Our investigation successfully identified pathogens likely associated with AFI and SNDs, underscoring the feasibility of retrieving near-complete genomes from RNA viruses. These findings offer promising prospects for advancing our understanding and control of infectious diseases, by facilitating detailed genomic analysis which is critical for developing targeted interventions and therapeutic strategies.

Potential use of high-resolution melting analyses for SARS-CoV-2 genomic surveillance.

The pandemic caused by COVID-19 and the emergence of new variants of SARS-CoV-2 have generated clinical and epidemiological impacts on a global scale. The use of strategies for monitoring viral circulation and identifying mutations in genomic regions involved in host interaction are important measures to mitigate viral dissemination and reduce its likely complications on population health. In this context, the objective of this work was to explore the potential of high-resolution melting (HRM) analysis combined with one-step real-time reverse transcription PCR in a closed-tube system, as a fast and convenient method of screening for SARS-CoV-2 mutations with possible implications on host-pathogen interactions. The HRM analyses allowed the distinction of the Gamma, Zeta, Alpha, Delta, and Omicron variants against the predecessors (B.1.1.28, B.1.1.33) of occurrence in Brazil. It is concluded that the molecular tool standardized here has the potential to optimize the genomic surveillance of SARS-CoV-2, and could be adapted for genomic surveillance of other pathogens, due to its ability to detect, prior to sequencing, samples suggestive of new variants, selecting them more assertively and earlier for whole genome sequencing when compared to random screening.

Emergence of Dengue Virus Serotype 2 Cosmopolitan Genotype, Brazil.

We used nanopore sequencing and phylogenetic analyses to identify a cosmopolitan genotype of dengue virus serotype 2 that was isolated from a 56-year-old male patient from the state of Goiás in Brazil. The emergence of a cosmopolitan genotype in Brazil will require risk assessment and surveillance to reduce epidemic potential.

Influence of SARS-CoV-2 inactivation by different chemical reagents on the humoral response evaluated in a murine model.

Viral inactivation for antibody induction purposes, among other applications, should ensure biosafety, completely avoiding the risk of infectivity, and preserving viral immunogenicity. ß-propiolactone (BPL) is one of the most used reagents for viral inactivation, despite its high toxicity and recent difficulties related to importation, experienced in Brazil during the SARS-CoV-2 pandemic. In this context, the main objectives of this work were to test different inactivation procedures for SARS-CoV-2 and to evaluate the induction of neutralizing antibodies in mice immunized with antigenic preparations obtained after viral treatment with formaldehyde (FDE), glutaraldehyde (GDE), peroxide hydrogen (H2O2), as well as with viral proteins extract (VPE), in parallel with BPL. Verification of viral inactivation was performed by subsequent incubations of the inactivated virus in Vero cells, followed by cytopathic effect and lysis plaques observation, as well as by quantification of RNA load using reverse transcription-quantitative real time polymerase chain reaction. Once viral inactivation was confirmed, cell culture supernatants were concentrated and purified. In addition, an aliquot inactivated by BPL was also subjected to viral protein extraction (VPE). The different antigens were prepared using a previously developed microemulsion as adjuvant, and were administered in a four-dose immunization protocol. Antibody production was comparatively evaluated by ELISA and Plaque Reduction Neutralization Tests (PRNT). All immunogens evaluated showed some level of IgG anti-SARS-CoV-2 antibodies in the ELISA assay, with the highest levels presented by the group immunized with FDE-inactivated viral antigen. In the PRNT results, except for VPE-antigen, all other immunogens evaluated induced some level of neutralizing anti-SARS-CoV-2 antibodies, and the FDE-antigen stood out again with the most expressive values. Taken together, the present work shows that FDE can be an efficient and affordable alternative to BPL for the production of inactivated SARS-CoV-2 viral antigen.

Searching for plant-derived antivirals against dengue virus and Zika virus.

BACKGROUND: The worldwide epidemics of diseases as dengue and Zika have triggered an intense effort to repurpose drugs and search for novel antivirals to treat patients as no approved drugs for these diseases are currently available. Our aim was to screen plant-derived extracts to identify and isolate compounds with antiviral properties against dengue virus (DENV) and Zika virus (ZIKV). METHODS: Seven thousand plant extracts were screened in vitro for their antiviral properties against DENV-2 and ZIKV by their viral cytopathic effect reduction followed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method, previously validated for this purpose. Selected extracts were submitted to bioactivity-guided fractionation using high- and ultrahigh-pressure liquid chromatography. In parallel, high-resolution mass spectrometric data (MSn) were collected from each fraction, allowing compounds into the active fractions to be tracked in subsequent fractionation procedures. The virucidal activity of extracts and compounds was assessed by using the plaque reduction assay. EC50 and CC50 were determined by dose response experiments, and the ratio (EC50/CC50) was used as a selectivity index (SI) to measure the antiviral vs. cytotoxic activity. Purified compounds were used in nuclear magnetic resonance spectroscopy to identify their chemical structures. Two compounds were associated in different proportions and submitted to bioassays against both viruses to investigate possible synergy. In silico prediction of the pharmacokinetic and toxicity (ADMET) properties of the antiviral compounds were calculated using the pkCSM platform. RESULTS: We detected antiviral activity against DENV-2 and ZIKV in 21 extracts obtained from 15 plant species. Hippeastrum (Amaryllidaceae) was the most represented genus, affording seven active extracts. Bioactivity-guided fractionation of several extracts led to the purification of lycorine, pretazettine, narciclasine, and narciclasine-4-O-ß-D-xylopyranoside (NXP). Another 16 compounds were identified in active fractions. Association of lycorine and pretazettine did not improve their antiviral activity against DENV-2 and neither to ZIKV. ADMET prediction suggested that these four compounds may have a good metabolism and no mutagenic toxicity. Predicted oral absorption, distribution, and excretion parameters of lycorine and pretazettine indicate them as candidates to be tested in animal models. CONCLUSIONS: Our results showed that plant extracts, especially those from the Hippeastrum genus, can be a valuable source of antiviral compounds against ZIKV and DENV-2. The majority of compounds identified have never been previously described for their activity against ZIKV and other viruses.